Mycobacterium tuberculosis is a tough bug to kill. The sterilization of a M. tuberculosis infection in[unreadable] a patient with active tuberculosis requires a minimum of 6 months of treatment with 3-4 drugs taken[unreadable] daily. This tenacity is unparalleled in any other human bacterial pathogen. M. tuberculosis has[unreadable] evolved strategies to survive in the face of drugs and the host immune responses. The current[unreadable] dogma in the field suggests that survival is the result of the bacteria's ability to enter into a drug[unreadable] tolerant state, where it is non-replicative and dormant, but clearly viable. If drugs are stopped early[unreadable] or the immune system is compromised, dormant bacteria can revive and go on to active disease.[unreadable] As part of the program project, we are dedicating our time and resources to find the "Achilles' heel"[unreadable] of the dormant bacteria using mycobacterial genetics. For this proposal, we have generated a high[unreadable] throughput methodology of specialized transduction, which can be used to disrupt any nonessential[unreadable] gene. In addition, we have used conditional expression and specialized transduction to[unreadable] develop CESTET, a method to prove essentiality and to study a mycobacterial cell's fate upon[unreadable] depletion of the gene product. In vitro and in vivo assays have been developed to screen for[unreadable] essential genes and persistence factors. The group has a proven track record of successfully[unreadable] solving structures having solved 70% of the Mtb structures in the PDB, and we will build on this[unreadable] success to solve the structures of the new essential or persistence targets. Using the structural[unreadable] information, virtual ligand screens will be performed to identify new inhibitors. Thus, the goal of this[unreadable] Project is to validate targets, solve their three dimensional structures, and propose novel inhibitors[unreadable] for further drug development to radically shorten the time to treat TB.